The fast axonal transport of neuronal organelles has been intensively studied using in vitro systems, and an abundance of primary sequence information has been produced for potential mechanochemical motors that could drive the movement of organelles. However, we still know very little about how organelle transport is controlled and coordinated in intact cells, and we are far from assigning specific motor proteins to particular roles in the cell. One major aim of this study is to determine whether the motor protein kinesin supports anterograde movement of all axonal organelles, or whether different classes of organelles employ different means of force generation. This question will be addressed by disrupting kinesin function in cultured peripheral neurons by introducing antibodies or antisense oligonucleotides into the cells and quantifying the effects on the transport of distinct, identifiable classes of organelles. The second major aim is to elucidate the nature and regulatory function of kinesin phosphorylation. Despite evidence indicating that both kinesin and the kinesin-binding protein kinectin are phosphorylated in vivo, we do not know whether kinesin's motor function or organelle binding are affected by phosphorylation, and the kinases responsible are unknown. These questions will be addressed using metabolic labeling of neuronal cultures, cell fractionation, and in vitro motility and binding assays. In order to frame reasonable hypotheses about how the regulation of motor proteins gives rise to coordinated organelle transport, it is first necessary to understand what kind of regulated organelle traffic actually occurs in intact cells. Thus, the third major aim of the study is to determine how organelle transport is modulated to meet specific physiological needs of the cell. Experiments will focus on the coordination of mitochondrial movement with axonal outgrowth and ATP consumption, and on the differences in organelle transport between axons and dendrites. These questions will be addressed by manipulating axonal outgrowth, quantifying organelle behavior, and determining the distribution and function of motor proteins in differentiated neurons containing dendrites in addition to axons. Because it provides a metabolic link between the periphery of neurons and the site of synthesis in the cell body, fast axonal transport is essential for the development and maintenance of all nerves; thus, the insight into its mechanism gained by the proposed study can be expected to contribute to our understanding of the development, defects, and diseases of the nervous system. In addition, since neuronal organelle transport must share many features with organelle traffic in other cells, information acquired in this system should provide insight into many other systems of intracellular motility.